Abstract: The present disclosure discloses a universal aptamer-based colloidal gold lateral flow test strip for detecting small-molecule substances and belongs to the fields of analytical chemistry, medicine, environment, food safety detection, nano-biosensing and the like. An AuNPs@poly-DNA probe is used for rapidly and sensitively capturing an aptamer, streptavidin sprayed in a test zone and streptavidin-biotin-DNAc sprayed in a control zone do not need to be changed, and another substance can be detected only by changing a nucleic acid chain part of the AuNPs@poly-DNA probe. A universal colloidal gold lateral flow test strip, which is rapid, sensitive and low in cost, has been developed. The test strip method for detecting small-molecule substances is simple, convenient and rapid, and can be used for detection at any time.